MOLECULAR AND MICROBIOLOGICAL APPROACHES FOR RAPID ETIOLOGICAL DIAGNOSIS OF SYSTEMIC MYCOSES

The review presents common laboratory methods for the diagnosis of the majority of invasive fungal diseases, i. e. candidosis, aspergillosis and cryptococcosis. Some studies reveal an increasing range of the infectious agents such as Trichosporon and endemic fungal pathogens like Histoplasma, Blastomyces and Coccidioides. The most common serological tests for antigen and antibody-detection in body fluids (serum, plasma, bronchoalveolar lavage ) are: indirect immunofluorescense, ELISA – Platelia (enzyme linked immunosorbent assay), latex-agglutination, immunodiffusion and molecular techniques like PCR and Real Time PCR. Not only current data on invasive fungal disease diagnostic methods are reviewed, but also studies on new biomarkers and recent discoveries in molecular diagnostics. New molecular approaches are needed to provide faster results .


INTRODUCTION
It is estimated that around 1.9 million patients worldwide develop invasive fungal disease (IFD) each year. These infections are associated with high mortality and economic burden due to hospitalizations. Тherefore, a fast and timely diagnosis is needed (1).
Systemic fungal diseases may cause mortality in immunocompromised individuals (oncology, chemotherapy, transplantation, catheterized or heart prosthetic and AIDS patients, patients in intensive care units (ICU), etc.) ( fig.1).

Fig.1 Schematic representation of the types of fungal infections (https://microdok.com/)
Routine microscopic and culture based methods are the classical ones, but diagnosis is provided in 48-72 hours (2, 3) ( fig.2, 3). For this reason, faster and reliable at the same time screening methods for early detection of pathogenic fungi are needed. (4,5). The majority of invasive fungal diseases are still caused by Candida (6) and Aspergillus species, but recent studies indicate an increasing incidence of other species, such as Cryptococcus and Trichosporon (7). There are endemic fungi in some areas, for example in the USA Histoplasma, Blastomyces and Coccidioides are reported (8).

MICROSCOPIC EXAMINATION
Microscopy of fungi can be done following appropriate staining -by Gram, or by fluorescent dyes, cotton blue, etc. The formation of germ tubes in C. albicans is a factor of virulence, which can be detected by the so-called Germination test (Fig 4). Microscopy and identification of Aspergillus is not easy and requires a guidance. Aspergillus spores are carried by a long conidiophore, and it originates from the so-called "foot cell" (Fig.5). On its top it ends with the so-called vesicle, which can vary in shape and size. From the surface of the vesicle conidia-forming cells called mettuli and phialides, begin, which can partially or completely cover the vesicle.

HISTOLOGY
Histological examination of biopsy materials for the presence of fungal elements is considered one of the main criteria for а proven fungal infection (Fig 6). Biopsy or surgical resection of pathological tissue are often definite signs for the diagnosis of an IFD). Timeliness and expedience are essential for early diagnosis.

CULTURE EXAMINATION
Sabouraud dextrose agar or chromogenic medium with color reaction during growth for 48-72 hours at 30 o С, the so called Chrom agar, is the most often used solid nutrient medium for the culture study. Following isolation of a fungal strain as a pure culture, biochemical identification follows. This can be achieved with the help of non-automated (commercial identification kits) or automated identification systems (VITEK2, Merlin, Phoenix, MALDI-Toff, Microscan, etc.). There are various biochemical identification kits, the most commonly used of which are Api 20 CAux and Auxacolor.

ANTIBODY DETECTION
The method that we use for determination of specific antibodies against Candida, Aspergillus and Cryptococcus is the indirect immunofluorescence (IIF). For performing it, it is necessary to prepare a microscopic slide from a culture suspension (   Another serological test for the detection of circulating antigens using a sample technique, is the latex agglutination (LA) test (BioRad). It is a qualitative test detecting the polysaccharide antigen, mannan (for Candida) and glucoronoxylomannan (Cryptococcus neoformans) in biological fluids (serum, BAL, cerebro-spinal fluid (CSF)). Thе procedure uses latex particles coated with monoclonal rat antibody directed against the fungal antigen (13). The particles react with the fungal antigen resulting in agglutination visible to the naked  .9) (14). Detection of Candida antigen by LA in body fluids is a reliable criterion for а systemic infection. LA tests for antigen detection in immunocompromised patients with cryptococcosis of the central nervous system (CNS) are even more reliable. The test has a very high sensitivity and specificity.

Fig. 9. LA test for yeast
A third method for antibody detection is immunodiffusion (ID). In our laboratory we have experience for searching antibodies against Aspergillus and endemic fungal pathogens such as Histoplasma, Blastomyces and Coccidioidesthe spill phenol agar for ID in a few petri plates. Immediately after spilling agar, the petri plate is placed in the refrigerator at 4 o C. After making wells with smooth edges, we drip: • Control serum (commercial kit) • Antigen (commercial kit) • Patient's serum Precipitation lines are sought following incubation in a wet camera at 25 o C for a few days (9).

MOLECULAR GENETIC METHODS FOR DIAGNOSIS
The non-culture based method is PCR -DNAdetection for the rapid diagnosis of infectionsamplification of a specific DNA-fragment by means of short, artificially synthesized oligonucleotides (primers) (15;16). They hybridize specifically with a particular region of the target DNA, with participation of Taq-polymerase (9). The following are the three reaction steps in one PCR cycle: 1. Denaturation of double-stranded DNA (dsDNA) to two single-stranded chains at 95°C (also called DNA melting) 2. Binding of primers to their complementary regions in the single-stranded ssDNA chains (also called annealing of primers) 3. Amplification of new DNA at each end in the 5'-3' direction, using DNA Taq polymerase in the presence of deoxynucleotides (dATP, dTTP, dCTP and dGTP) to build new daughter DNA frangments.

REAL-TIME PCR
Real-time PCR allows tracking of DNA amplification at the time it occurs, not at the end of the reaction, as in conventional PCR (17). In most eukaryotes, coding rDNA genes and spacers are found in tandem repeats separated by non-transcribable DNA, called "no"-transcribed spacer (NTS). The coding regions of 18S, 5.8S and 28S nuclear ribosomal DNA (rDNA genes) are relatively conserved among fungi and provide a molecular basis for establishing phylogenetic links. Between the coding regions are: the internal transcribed spacer 1 and 2 -regions, respectively ITS1 and ITS 2. The ITS region is perhaps the most widely sequenced DNA region in fungi. Universal fungal primers are used to amplify the entire internal transcribable ITS spacer. The ITS region, located between 18 S and 28S ribosomal DNA, is divided into two (ITS 1 and ITS2), and between them the preserved region 5.8S is situated (Fig.10) Amplification techniques offer increased sensitivity over traditional staining and culture methods but may report positive results in asymptomatic individuals because of colonization (18; 19). Real time PCR has advantages as speed, enhanced sensitivity and wider range of detectable organisms, which cannot be cultured (20). Double-labeled hybridization probes can be used. The probe is an oligonucleotide that is labeled with a fluorescent dye (reporter) at one end and a fluorescence quencher at the other end. The proximity of the extinguisher to the fluorescent dye suppresses the emission of the natural fluorescence by the fluorescent dye. In PCR, the hybridization probe binds to a specific region of the template DNA. Thus, there is a directly proportional correlation between the increased fluorescent signal and the amount of the PCR product (Fig.11) Real-Time PCR can also be used for quantitative analysis -determination of the number of DNA molecules in the sample in real time -to distinguish colonization from infection, because some medically important fungi are opportunistic pathogens. They are part of the normal human microflora.

Empirical therapy
Target therapy

GENERAL CONSIDERATIONS
The different methods for laboratory diagnosis of IFD vary in specificity and sensitivity (21 (26).

CONCLUSIONS
Genetic methods perform higher specificity than serological ones. They can significantly reduce detection time, but there are still no fully standardized protocols for isolating fungal DNA and performing PCR (27;28). Standartization is necessary for their introduction in the routine practice in mycological laboratories. Problems with the avoidance of contamination of clinical materials and with the development of methodologies to distinguish infection from contamination are to be resolved (29). Мetaanalysis confirms that if both PCR and Elisa are consistently negative, then this is sufficient to rule out invasive aspergillosis (30). Clinical mycology, unlike bacteriology, has a slower pace of development. The use of serological tests for antigens and antibodies, as well as the introduction of molecular techniques make non-cultural methods essential for the prediction of IFD. None of these tests, however, can be used as a stand-alone definitive diagnostic test. Compliance with clinical data, imaging studies, etc is essential (1). Laboratory diagnosis of invasive fungal disease has to be used in conjunction with other diagnostic procedures such as culture technique, histological examination of biopsy samples, computed tomography (CT) imaging (31,32). Further research is needed to establish the benefits of PCR for fungal detection in routine practice (26).

ACKNOWLEDGEMENT
This work was supported by Operative Program "Science and Education of Intelligent Growth", project BG05M2OP001-1.002-0001-C04.